Despite a large and increasing research effort aimed at growing functional connective tissues in vitro, with few exceptions they lack the required functionality because the tissue growth is limited and the resulting material/mechanical properties are inadequate for in vivo applications. While chemical and mechanical signals, alone or in combination, can indeed lead to improved tissue growth as measured by material/mechanical properties, the current paradigm of applying these signals at fixed levels for the entire period of tissue growth is almost certainly suboptimal. The vision for the proposed project is to develop a rational basis for optimizing in vitro tissue growth via combined chemical and mechanical stimulation that does not require knowledge of the signal generation and transduction mechanisms, which are so daunting in complexity that a "first principles" model is unrealizable for the foreseeable future. It is inspired by a systems biology approach recently advanced by Janes and Lauffenburger (Janes, Kelly et al. 2004) based on principal component analysis and discriminate partial least squares regression that we will extend to determine a statistical relation between long-term "downstream" cell responses of interest (collagen and elastin production and associated tissue mechanical properties) and a subset of short-term "upstream" intracellular signals (i.e. phosphorylated proteins) that are generated due to extracellular stimuli. Having determined this "signal-response reduction" from a series of long-term step-response experiments on cells subject to various concentrations of chemical (e.g. TGF-b1) and mechanical (stretching) stimulation, an "experimental steepest descent" strategy will then be implemented to drive the cells as they grow a tissue to produce the maximum amount of collagen and elastin, which should maximize the tissue strength and modulus while imparting elasticity. This will be accomplished by periodically performing short-term step-response ("interrogation") experiments in situ and using the predetermined reduction to predict the optimal combination of TGF-b1 concentration and stretching conditions for the subsequent incubation period, after which the tissue is again interrogated and the optimal conditions are again updated, and so on. If successful, this paradigm would dramatically change the field of tissue engineering and likely elucidate signaling pathways that lead to deposition of extracellular matrix components. PUBLIC HEALTH RELEVANCE: Few attempts to grow functional tissue replacements have succeeded. In order to grow stiffer and stronger tissues, a rationale is needed to choose the incubation conditions. In this project, high- throughput cell signaling data will be obtained periodically during the incubation to adjust the incubation conditions so that tissue growth is improved.